Instrumentation Core. All of the projects in this program project grant will rely heavily upon collection of cw-EPR and double electron-electron resonance (DEER) data from site-directed spin-labeled proteins. This will necessitate users having access to EPR spectrometers that are set-up and optimized for many different kinds of experiments. The investigators on this program project grant will have access to three different EPR instruments. The first is a Bruker EMX system that operates at X-band microwave frequency. This instrument is the work-horse for initial characterization of spin-labeled samples, for recording final publication quality data at X-band, and for making NiEDDA (or O2) side-chain accessibility measurements. There are a wide range of resonators available for this instrument including standard TM110 and TEi02 mode cavities, a Medical Advances loop-gap resonator, a Bruker dielectric resonator (ER 4118X-MD-5) and a Bruker split-ring resonator (ER 4118X-MS-2W). The second instrument is also a Bruker EMX that has both an X- and a Q-band microwave bridge. This instrument is normally set up to operate at Q-band using a Bruker ER 5106QT cavity. However, it can be quickly converted to X-band operation when the work-load is too great on the first instrument. These systems can be set up to do temperature controlled experiments (from liquid nitrogen to physiological) using different carrier gases (N2, air, O2) and the cavity/resonator that will be best suited to the samples being investigated. Dr. Cobb will be available to set up and optimize both of these instruments for all of the samples being studied. He has extensive experience in this capacity having been in charge of the two EPR spectrometers in the Beth lab for the past decade and in his role of directing the fluorescence spectroscopy facility located in the Department of Molecular Physiology & Biophysics. The third instrument that will be routinely used is the new Bruker EleXsys E-580 FT-EPR spectrometer that was installed adjacent to the Beth and Hustedt laboratories in the Department of Molecular Physiology & Biophysics in early May, 2006. This instrument is set up to do temperature controlled pulsed EPR X-band experiments (liquid He to physiological) on spin-labeled samples using either a dielectric (ER 4118X-MD-5) resonator or a split ring (ER 4118X-MS-2W) resonator. Dr. Cobb obtained extensive training from Bruker during the installation of this instrument and it is now being utilized to collect DEER data on spin-labeled protein samples on a routine basis. He will not only have responsibility for setting this new instrument up for specific experiments, but he will also be responsible for training new users including students and post-docs. The single new capability that is requested on this program project grant is funding for a pulsed Q-band microwave bridge and resonator for making DEER measurements on the full range of spin-labeled samples to be investigated. This equipment will permit the acquisition of experimental DEER data from selected samples at a second, higher microwave frequency. One of the greatest challenges in analyzing experimental DEER data in terms of multiple average distances and distance distributions is determining how unique the multimodal fits are. By requiring that the experimental data simultaneously fit the data at two well-separated microwave frequencies, the reliability of the recovered parameters will be significantly increased. The value of similar multi-frequency analysis approaches has been demonstrated in previous work from this applicant group (e.g. Hustedt et al., 1997) and by the work on side-chain dynamics by Hubbell, Freed and coworkers (e.g. Barnes etal., 1999). All of the existing instruments will be available to users on this program project grant by on-line sign-up on a 24 hour/7 day basis via keyed access.